1. Field of the Invention
The present invention relates to a pre-treatment kit for saliva and a pre-treatment method for saliva using the kit, which are used for identification and quantitation of a bacteria belongs to mutans streptococci that is one of cariogenic bacteria in human saliva, by the immunochromatographic method utilizing an antigen-antibody reaction.
2. Description of the Conventional Art
It is known that the presence of mutans streptococci in a human oral cavity is closely related to the generation of dental caries. If the presence or absence or the amount of mutans streptococci in the human oral cavity could be examined simply, it would become possible to grasp a disease risk or the current disease state. Thus, an extremely large number of people should possibly enjoy such benefits.
Hitherto, an examination utilizing an antigen-antibody reaction has been carried out for the examination of bacteria. For example, an enzymatic antibody method is a method for identification and quantitation by a color development density using an enzyme. However, this method requires not only a special washer and a complicated and precise operation for dealing with an antibody or a sample but also an incubator for achieving an enzymatic reaction. Further, a fluorescent antibody method is a method for labeling an antibody with a fluorescent dyestuff to dye specifically an antigen having reacted with the antibody. However, this method is not general because it requires a fluorescent microscope as an assay instrument.
For these reasons, a number of methods utilizing an antigen-antibody reaction simply have been proposed. For example, assay methods utilizing chromatography, as disclosed in U.S. Pat. Nos. 5,591,645, 4,855,240, 4,435,504 and 4,980,298, Japanese Patent Laid-Open Nos. 145459/1986 and 160388/1994, and so on, are a method that is superior in simplicity, upon which the presence or absence or the amount of an antigen can be known only by incorporating a collected body fluid into a test solution containing the antibody for the purpose of the identification and quantitation, and then infiltrating it into a test appliance. These methods are called generally an immunochromatographic method. In such methods, a specific antibody that attaches only to a target antigen (this antibody will be simply referred to “specific antibody”, hereinafter) is infiltrated into one end of a porous membrane (a pore diameter: several tens μm) such as nitrocellulose, and another specific antibody similarly attaching only to a specific antigen is infiltrated in a stripe form into the middle of the porous membrane and fixed to the porous membrane. The specific antibody infiltrated into one end of the porous membrane is colored with particles of, e.g., colloidal gold in advance. When a sample solution is infiltrated into one end of the porous membrane where the specific antibody is present, so far as an antigen that is reactive with the specific antibody is present in the sample solution, the antigen is coupled with the specific antibody and moves in the state of attaching the coloring particles by the capillary action in the porous membrane toward the opposite end to the side into which the sample solution is infiltrated. During the movement, when the antigen passes through a portion where another specific antibody is fixed in a stripe form, the specific antibody on the porous membrane traps the antigen, whereby a stripe-like blot appears on the porous membrane. Thus, the presence of the target antigen in the sample and its amount can be known.
If such a technology were applied, it would appear possible to undergo the identification and quantitation of the above-described intraoral mutans streptococci. However, actually, such has not yet been put into practical use because of the presence of a problem as described below. That is, a sample that can be used in the immunochromatographic method should be able to pass in principle through the porous membrane by the capillary action. However, since a major sample that is used for the examination of intraoral bacteria such as mutans streptococci is saliva, a highly viscous substance that is called mucin present in the saliva clogs pores of the porous membrane. Also, the mucin acts to aggregate epithelium-attaching cells present in the saliva, which have come off and dropped from an oral mucosa surface. Accordingly, such a substance clogs the pores of the porous membrane so that the mutans streptococci cannot pass through the porous membrane.
Also, in addition to the matter of mucin, there is a problem to make the assay of mutans streptococci becomes difficult. That is, the objective mutans streptococci are bacteria having a diameter of about 1 μm in terms of a single body. However, since the mutans streptococci are streptococci, from ten to twenty or more of them are often chained with each other, which causes to hinder the movement within the porous membrane. Moreover, the mutans streptococci generate viscous glucan from sucrose in foods, whereby they are often aggregated vigorously to each other. Still further, the chaining and aggregation of the mutans streptococci not only cause clogging of the porous membrane but also reduce the surface areas of the streptococci and influence the number of antigens present on the surfaces of the mutans streptococci, resulting in lowering in the assay precision.